Favazza M, Lerho M, Houssier C
Laboratoire de Chimie Macromoléculaire et Chimie Physique, Université de Liège, Sart-Tilman, Belgium.
J Biomol Struct Dyn. 1990 Jun;7(6):1291-300. doi: 10.1080/07391102.1990.10508566.
Histone H5 has been labelled with fluorescein isothiocyanate (FITC) with particular attention to the reaction conditions (pH, reaction time and input FITC/H5 molar ratio) and to the complete elimination of non-covalently bound dye. We preferred to use reaction conditions which yielded non-specific uniform labelling rather than specific alpha-NH2 terminal labelling, in order to obtain higher sensitivity in further studies dealing with the detection of perturbation at the binding sites of H5 on DNA. FITC-labelled H5 was further characterized by absorption and circular dichroism spectroscopy, and the fluorescein probe titrated in the 4-8 pH range. The structural integrity of H5 was found to be preserved after labelling. The positive electrostatic potential of the environment in which the FITC probe is embedded in the arginine/lysine-rich tails of H5 is believed to be responsible for the drop of pK of 1 unit found for H5-FITC as compared to free FITC. For the globular part of H5, the pK of covalently-bound FITC was only slightly lowered; this is a consequence of the much lower content in positively-charged amino-acid side chains in this region.
组蛋白H5已用异硫氰酸荧光素（FITC）进行标记，特别关注反应条件（pH值、反应时间和FITC/H5摩尔比）以及非共价结合染料的完全去除。为了在进一步研究H5与DNA结合位点的扰动检测中获得更高的灵敏度，我们倾向于使用能产生非特异性均匀标记而非特异性α-NH2末端标记的反应条件。通过吸收光谱和圆二色光谱对FITC标记的H5进行了进一步表征，并在4-8的pH范围内对荧光素探针进行了滴定。发现标记后H5的结构完整性得以保留。据信，FITC探针嵌入H5富含精氨酸/赖氨酸的尾部所处环境的正静电势，是导致H5-FITC的pK值相较于游离FITC下降1个单位的原因。对于H5的球状部分，共价结合的FITC的pK值仅略有降低；这是该区域带正电荷的氨基酸侧链含量低得多的结果。
